Anti nucleolin antibody

Manufactured by Abcam

Sourced in United Kingdom, China, United States

Anti-nucleolin antibody is a laboratory reagent used to detect and quantify the presence of nucleolin, a multifunctional protein involved in various cellular processes. This antibody can be used in techniques such as Western blotting, immunohistochemistry, and immunoprecipitation to investigate the expression and localization of nucleolin in biological samples.

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8 protocols using anti nucleolin antibody

Immunofluorescence Staining Protocol

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Dulbecco’s modified minimal essential medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), Trypsin-EDTA solution, 100× penicillin–streptomycin–l-glutamine solution, anti-RFP antibody, Alexa 647-conjugated secondary antibody, SlowFade Gold antifade reagent, and a LIVE/DEAD fixable yellow dead cell stain kit were purchased from Thermo Fisher (Waltham, MA USA). Puromycin solution and G418 solution were obtained from InvivoGen (San Diego, CA, USA). Hygromycin B solution was purchased from Enzo Life Science (Farmingdale, NY, USA). Anti-ORF57, anti-K8, and anti-K8.1 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-LANA and anti-lamin A/C antibodies were purchased from Millipore-Sigma (St. Louis, MO, USA). Anti-nucleolin antibody was purchased from Abcam (Cambridge, UK). Anti-K-Rta antibody was described previously (68 (link)). Monoclonal anti-ORF52 antibody was a generous gift from Fanxiu Zhu (Florida State University). All other chemicals were purchased from Millipore-Sigma (St. Louis, MO, USA) unless otherwise stated.

Nakajima K.I., Guevara-Plunkett S., Chuang F., Wang K.H., Lyu Y., Kumar A., Luxardi G., Izumiya C., Soulika A., Campbell M, & Izumiya Y. (2020). Rainbow Kaposi's Sarcoma-Associated Herpesvirus Revealed Heterogenic Replication with Dynamic Gene Expression. Journal of Virology, 94(8), e01565-19.

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Nucleolin Localization in MCF-7 Cells

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MCF-7 cells were grown on confocal observation dishes for 18 h. The culture medium was removed and cells fixed by 100 μL 4% formaldehyde and incubated on ice for 5 min. To stain cell surface nucleolin, cells were pre-incubated with blocking buffer (5% bovine serum albumin in PBS) for 1 h, washed twice with PBS, and then incubated with anti-nucleolin antibody (Abcam) at 4 °C overnight. Cells were then washed twice with PBS and incubated with Alexa Fluor 647-conjugated anti-rabbit immunoglobulin G (IgG, Life technologies) (1:1000) for 1 h at 37 °C. Cells were washed three times and then incubated with 550 nM FAM-FAN-1524dI for 30 min at 37 °C. The cells were again washed three times in PBS and then permeabilized with 0.5% (v/v) Triton X-100 in PBS buffer for 10 min. Cells were stained with 5 μg/ml Hoechst 33342 (50 μL per well) for 30 min and observed under an A1Rsi confocal microscope (Nikon Instruments Inc.). Confocal images were obtained using NIS-Elements and Bitplane imaris software (Nikon Instruments Inc.).

Fan X., Sun L., Wu Y., Zhang L, & Yang Z. (2016). Bioactivity of 2′-deoxyinosine-incorporated aptamer AS1411. Scientific Reports, 6, 25799.

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Immunofluorescence Analysis of DNA-RNA Hybrids

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Immunofluorescence using S9.6 antibody (Kerafast INC.) was performed as previously described (21 (link)). Cells were incubated with primary antibodies, anti-DNA-RNA hybrid [S9.6] antibody (1:500, Kerafast INC.), anti-nucleolin antibody (1:1000, Abcam Cat # ab22758), anti-PARP1 antibody (1:1000, Abcam Cat # ab32138 and 1:500 Cat # ab191217), anti- XRCC1 antibody (1:1000, Cell Signaling Technology Cat #27350), anti-DNA Polymerase beta antibody (1:1000, Abcam Cat # ab26343), anti-CYCLIN A antibody (1:200, Sigma Cat #C4710), and anti-RPA70/RPA1 antibody (1:50, Cell Signaling Technology Cat #2267S) followed by incubation in secondary antibodies Alexa Fluor 488 goat anti-mouse (1:1000, Invitrogen Cat #A11034) or Alexa Fluor 594 goat anti-rabbit (1:1000, Invitrogen Cat #A11005). DNA was stained with DAPI. Images were captured at 40X magnification with a Zeiss Confocal Microscope (Zeiss LSM 700). Fiji software (version: 2.0.0-rc-69/1.52i, open source image processing software) used for analysis of images. As a control, cells were treated with 10 units of RNase H enzyme (New England Biolabs Cat # M0297L), prior to staining with S9.6 antibody.

Safari M., Litman T., Robey R.W., Aguilera A., Chakraborty A.R., Reinhold W.C., Basseville A., Petrukhin L., Scotto L., O'Connor O.A., Pommier Y., Fojo A.T, & Bates S.E. (2021). R-Loop–Mediated ssDNA Breaks Accumulate Following Short-Term Exposure to the HDAC Inhibitor Romidepsin. Molecular Cancer Research, 19(8), 1361-1374.

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Quantifying Nucleolin Expression

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5.0 × 10⁴ HUVECs were suspended in phosphate buffer at pH 7.4 in the presence of tTF-Cy5.5, tTF-AS1411-Cy5.5 or tTF-AS1411-Cy5.5 (20 μg tTF for each formulation) and incubated with an anti-nucleolin antibody (1:1000, Abcam, catalog No. ab129200, Shanghai, China) for 30 min at 37 °C. The cells were washed three times with PBS and then resuspended in 600 μL PBS for flow cytometry analysis (Becton and Dickinson C6, Franklin Lake, NJ, USA).

Li B., Wei J., Di C., Lu Z., Qi F., Zhang Y., Leong W.S., Li L., Nie G, & Li S. (2020). Molecularly engineered truncated tissue factor with therapeutic aptamers for tumor-targeted delivery and vascular infarction. Acta Pharmaceutica Sinica. B, 11(7), 2059-2069.

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Tumor Targeting with DOX@UiO-66/Py-PGA-PEG-F3

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MDA-MB-231 tumor-bearing mice were injected with DOX@UiO-66/Py–PGA-PEG-F3 and DOX@UiO-66/Py–PGA-PEG (5 mg of DOX/kg of mouse body weight) and sacrificed at 2 h p.i. MDA-MB-231 tumors from each injection group were frozen cut into 6-μm slices and incubated with 5 μg/mL antinucleolin antibody (Abcam, Cat # ab22758) at room temperature for 1 h. After that, tumor slices were rinsed repeatedly by PBS and incubated with 2 μg/mL FITC-labeled goat antirabbit IgG (Jackson Immunoresearch) for 1 h at room temperature. Immunostained tumor slices were examined under Nikon A1 confocal microscope with a magnitude of 200×.

Chen D., Yang D., Dougherty C.A., Lu W., Wu H., He X., Cai T., Van Dort M.E., Ross B.D, & Hong H. (2017). In Vivo Targeting and Positron Emission Tomography Imaging of Tumor with Intrinsically Radioactive Metal–Organic Frameworks Nanomaterials. ACS nano, 11(4), 4315-4327.

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Radiolabeled Antibody Conjugate Synthesis

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Zirconium chloride (ZrCl₄), terephthalic acid (BDC), benzoic acid (BA), 1-pyrenemethylamine hydrochloride, triethylamine (TEA), poly(γ-glutamic acid) (γ-PGA), 1-ethyl-3-(3-(dimethylamino)-propyl) carbodiimide hydrochloride (EDC·HCl), and 37% HCl were all purchased from Sigma-Aldrich (St Louis, MO). ⁸⁹Zr-oxolate (in 1 M oxalic acid) was acquired from University of Wisconsin Cyclotron group. Doxorubicin hydrochloride and tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl) were ordered from Thermo Fisher Scientific (Fair Lawn, NJ). Amine-PEG-Mal (i.e., amine PEG maleimide, MW: 5 kDa) was purchased from Creative PEGworks (Chapel Hill, NC). Antinucleolin antibody and anti-β-actin antibody (conjugated with horseradish peroxidase [HRP] for Western blotting) were both purchased from Abcam (Cambridge, MA, USA). Secondary HRP antibodies were purchased from Jackson ImmunoResearch (St. Louis MO, USA). Chelex 100 resin (50–100 mesh) was purchased from Sigma-Aldrich. Buffers used in this study were prepared from Millipore-grade water and pretreated with Chelex 100 resin to ensure that the aqueous solution was free of heavy metals. Size exclusive PD-10 columns were purchased from GE Healthcare (Piscataway, NJ). All other chemicals were purchased from Thermo Fisher Scientific.

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Immunofluorescence Quantification of DNA-RNA Hybrids

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Immunofluorescence using S9.6 antibody (Kerafast Inc.) was performed as previously described (21 (link)). Cells were incubated with primary antibodies, anti-DNA–RNA hybrid (S9.6) antibody (1:500, Kerafast Cat # ENH001), anti-nucleolin antibody (1:1,000, Abcam Cat # ab22758), anti-PARP1 antibody (1:1,000, Abcam Cat # ab32138 and 1:500 Cat # ab191217), anti-XRCC1 antibody (1:1,000, Cell Signaling Technology Cat #27350), anti-DNA Polymerase beta antibody (1:1,000, Abcam Cat # ab26343), anti-CYCLIN A antibody (1:200, Sigma Cat #C4710), and anti-RPA70/RPA1 antibody (1:50, Cell Signaling Technology Cat #2267S) followed by incubation in secondary antibodies Alexa Fluor 488 goat anti-mouse (1:1,000, Invitrogen Cat #A11034) or Alexa Fluor 594 goat anti-rabbit (1:1,000, Invitrogen Cat #A11005). DNA was stained with DAPI. Images were captured at ×40 magnification with a Zeiss Confocal Microscope (Zeiss LSM 700). Fiji software (version: 2.0.0-rc-69/1.52i, open source image processing software) used for analysis of images. As a control, cells were treated with 10 U of RNase H enzyme (New England Biolabs Cat # M0297L), before staining with S9.6 antibody.

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Visualizing Cellular Uptake of Targeted Molecules

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HUVECs were maintained in confocal microscopy dishes (2 cm diameter; 5.0 × 10³/dish, Nunc, catalog No. 801002, Shanghai, China.) and cultured in F12K media (WISENT) supplemented with 10% FBS (WISENT) and 100 ng/mL VEGF (Beyotime). After 24 h, the media was discarded and cells were washed three times with phosphate buffer saline. Cy5.5-labeled tTF, Cy5.5-labeled AS1411 or Cy5.5-labeled tTF-AS1411 was then added to cells that were pretreated with anti-nucleolin antibody (Abcam) or not. After 30 min, the cells were washed three times with phosphate buffer saline. DiO (Solarbio, catalog No. D5840, Beijing, China) dye was used to stain the cell membranes and Hoechst 33342 (Solarbio, catalog No. B8040, Beijing, China) was used to stain cell nuclei. The FV1000-IX81 confocal microscope (Olympus, Tokyo, Japan) was used to acquire and analyze images.

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